Study On The Molecular Mechanism Of Sodium-dependent Inorganic Phosphate Transporter PiT2 In Neuronal Development

PiT2 is a member of the type-III inorganic phosphate(Pi)transporters,encoding by SLC20A2.PiT2 is a transmembrane protein composed of 652 amino acids.Based on experimental evidence and bioinformatics prediction,PiT2 is proposed to comprise 12 transmembrane domains,6 intracellular regions and 5 extracellular regions with extracellular N-and C-terminal tails.PiT2 is composed of mainly two PD domains(N-PD-1131 and C-PD-1131)and a large intracellular loop between them(the seventh loop composed of 248 amino acids,known as loop7).PD domain also plays an important role in maintaining transport function,and loop regions in PD domain,such as 67-91,107-141,517-530 amino acid residues are required for amphotropic murine leukemia virus(A-MuLV)binding.The exact functions of loop7 domain are still poorly understood.It was found that the sodium phosphate co-transport and retroviral recognition function were not affected when the loop7 domain was deleted.PiT2 is extensively expressed in the nerve system.What is the role of loop7 domain in the nervous system?To investigate possible functions of the loop7 domain in the nervous system,immunofluorescence assays of Neuro2A cells transfected with PiT2 interference or loop7 deletion vectors wre performed.Cofocal images showed suppression PiT2 expression or loop7 deletion induced a significant decrease in neurite length.Furthermore most of the loop7 deletion mutants were found in cytoplasm,and more mutant proteins aggregated in a specific region of cytoplasm.In order to further understanding of interacting proteins of PiT2 participates in regulating subcellular localization and neurite outgrowth,we performed yeast two hybrid screening using residues 235-482(loop7 domain)as bait.In the determination of bait protein showed non-toxicty and can not activate reporter genes,human fetal brain cDNA Library was screened under high stringency conditions,35 positive clones were obtained from screening.After sequenced the cDNA insert and Blast in the Genebank,two candidate interaction proteins were found.Loop7 domain of PiT2 might interact with microtubule-associated protein 1B,PIP3-dependent Rac exchanger P-Rex1,LIM domain only 4 and erythrocyte membrane protein band 4.2.The interaction of PiT2 with MAP1B was confirmed by co-immunoprecipitation and immunofluorescence co-localization analysis.Alanine-scanning mutagenesis of the loop7 domain demonstrated that residues 386-390(YTCYT)contribute to interaction with MAP1B,mutations of MAP IB binding site in the loop7 domain decreased the length of neurites in Neuro2A cells.Compared with undifferentiated Neuro2A cells,PiT2 proteins immune-precipitating with LC1 were roughly doubled in the differentiated Neuro2A cells.These results indicate that PiT2 might participate in the regulation of neuronal outgrowth by interacting with MAP 1B.Then three PiT2 mutants from the primary brain calcification family were introduced into neurite outgrowth assay.Following induction of differentiation,overexpression of Pi transport function deficient mutant PiT2-S601W(a missense mutation in C-PD domain)and mutant PiT2-V507Efs*2(only deleted C-PD domain)did not affect the neurite outgrowth in Neuro2A cells.Overexpression of mutant PiT2-R254*(deleted loop7 and C-PD domain)as similar to mutants PiT2-386-390A and PiT2-Aloop7,significant decreased length of neurites in Neuro2A cells.The interaction of PiT2 with P-Rex1 was confirmed by co-immunoprecipitation.In the study of the physiological significance of the interaction between PiT2 and P-Rexl,it was found that absence of P-Rexl induced a decrease in neurite length in the process of differentiation.This is consistent with the phenotypic effects of PiT2 on neurite outgrowth.Furthermore,Overexpression of P-Rexl in Neuro2A cells increase the neurite length,P-Rexl could promote neurite extension of Neuro2A cells.GTPase activity ssays showed that PiT2 and P-Rexl have similar regulatory patterns to small G protein Rac,which can promote the activity of Rac in the differentiation of Neuro2A cells.Overexpression of PiT2 and suppression P-Rex1 expression at the same time decreased the activity of Rac and the length of neurites in Neuro2A cells.Therefore,the regulation of PiT2 on neurite extension and Rac avtivition of Neuro2A cells might be implemented by the interaction with P-Rex1.In summary,we found that loop7 is a newly-identified functional domain,and is implicated in the differentiation of Neuro2A cell.We also found two interacting proteins(MAP1B and P-Rex1)of PiT2 involved in the regulation of neural development.PiT2 might regulate neurite extension of Neuro2A cells through interaction with MAP1B or P-Rex1.These findings might provide a novel mechanism that PiT2 regulates neural outgrowth,a process that might contribute to neuronal development.